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骨干人才

杨　辉

杨辉，1985年8月生，博士，中国科学院脑科学与智能技术卓越创新中心研究员，博士生导师。 2007年于上海交通大学获得生物技术学士学位。2012年于中国科学院上海生命科学研究院获得发育生物学博士学位。2012年至2014年在美国麻省理工学院Whitehead研究所从事博士后研究工作。同年回国加入中国科学院脑科学与智能技术卓越创新中心工作，任非人灵长类疾病模型研究组组长。2015年获得国家自然科学基金优秀青年科学基金资助，同年入选“上海市青年拔尖人才”；2018年获得“上海市自然科技技术一等奖”、中科院上海分院“杰出青年科技创新人才奖”；2019年获得国家自然科学基金杰出青年科学基金资助,同年获得何梁何利科学与技术进步奖、中源协和生命医学奖、药明康德生命化学研究奖和上海市科技系统青年五四奖章个人奖。 “单碱基基因编辑造成大量脱靶效应及其优化解决方法”研究成果入选“2019年中国生命科学十大进展”。

    主要研究方向及内容

    一、研究方向：基因编辑技术的应用研究

    目前已知的遗传性疾病有七千余种，但绝大部分缺乏有效的治疗药物和方法。随着新型基因编辑技术CRISPR-Cas9、单碱基编辑等的出现和快速发展，为这些疾病的治疗带来了曙光。然而基因编辑技术的脱靶效应和编辑效率等问题，阻碍了基因编辑技术在体细胞基因治疗的有效、精准应用。同时基因修饰动物模型在疾病的研究和治疗中扮演着重要角色，而采用基因编辑技术获得基因修饰动物模型的效率仍有待提高，限制了该技术的广泛应用。因此，本课题组将围绕新型基因编辑技术的开发、安全性检测，非人灵长类动物模型研发和疾病治疗中的应用开展工作。  

    二、研究内容

    1．基因编辑技术安全性评价工具

    (1)本课题组建立了一种被命名为GOTI（Genome-wide Off-target analysis by Two-cell embryo Injection）的新型脱靶检测技术，并使用该技术发现近年来兴起的单碱基编辑技术有可能导致大量无法预测的脱靶，因而存在严重的安全风险。此研究显著提高了基因编辑技术脱靶检测的敏感性，并且可以在不借助于任何脱靶位点预测技术的情况下发现之前的脱靶检测手段无法发现的完全随机的脱靶位点，为基因编辑工具的安全性评估带来了突破性的新工具，有望成为新的行业检测标准。(Zuo et al., Science, 2019)

    (2)通过分析转录组数据证明单碱基编辑工具会导致转录组范围内的脱靶效应，并且脱靶产生的单核苷酸突变与目的编辑位点没有序列相似性，而主要是由于DNA单碱基编辑器的脱氨酶APOBEC1 (BE3)和TadA (ABE)所导致的。此外，团队还发现很高比例的RNA脱靶发生在癌基因和抑癌基因上，如果用于临床治疗有较大的致癌风险。该项研究发现了单碱基编辑工具还存在无法预测的RNA脱靶，加强了世人对单碱基编辑工具的安全性的审视。(Zhou et al., Nature, 2019) 胞嘧啶单碱基编辑器的脱靶是由其上的脱氨酶产生的。脱氨酶利用自身的ssDNA和RNA结合能力，携带Cas9蛋白在基因组中随机与ssDNA和RNA结合，并且利用自身催化活性将C突变为T，从而造成单碱基基因编辑工具在基因组和转录组范围内完全随机无法预测的脱靶效应。通过在CBE的脱氨酶APOBEC1上引入突变，以此消除ssDNA和RNA的结合能力。在23个CBE突变体中， 4个突变体不影响基因编辑效率，通过DNA和RNA的脱靶检测后获得的3个突变体BE3R126E、BE3R132E和YE1-BE3能够显著降低DNA和RNA的脱靶效应。为了进一步提高YE1-BE3编辑效率，研究者随后又在突变体基础上增加标签和核定位序列（FNLS）。优化后的单碱基编辑工具YE1-BE3-FNLS在保证高保真的情况下，显著提高了基因编辑效率，从而成为既安全又高效的新的基因编辑工具。(Zuo et al., Nature Methods, 2020)  

    2．新型基因编辑工具开发

    胞嘧啶单碱基编辑器的脱靶是由其上的脱氨酶产生的。脱氨酶利用自身的ssDNA和RNA结合能力，携带Cas9蛋白在基因组中随机与ssDNA和RNA结合，并且利用自身催化活性将C突变为T，从而造成单碱基基因编辑工具在基因组和转录组范围内完全随机无法预测的脱靶效应。通过在CBE的脱氨酶APOBEC1上引入突变，以此消除ssDNA和RNA的结合能力。在23个CBE突变体中， 4个突变体不影响基因编辑效率，通过DNA和RNA的脱靶检测后获得的3个突变体BE3R126E、BE3R132E和YE1-BE3能够显著降低DNA和RNA的脱靶效应。为了进一步提高YE1-BE3编辑效率，研究者随后又在突变体基础上增加标签和核定位序列（FNLS）。优化后的单碱基编辑工具YE1-BE3-FNLS在保证高保真的情况下，显著提高了基因编辑效率，从而成为既安全又高效的新的基因编辑工具。(Zuo et al., Nature Methods, 2020)

    3．新型基因编辑工具在多种疾病应用

    (1)与Cas9介导的基因敲除技术相比，Cas13d介导的基因沉默不会改变基因组DNA，因此这种基因沉默是可逆的，从而对一些后天性疾病（如因不良生活习惯导致的高血脂等后天代谢性疾病）的治疗更有优势。其中Cas13d家族的CasRx蛋白由于体积小，效率高，被认为是在未来应用中最具有优势的Cas13蛋白。此前的工作都在细胞水平证明了CasRx的高效性和特异性，为临床提供了可能性，分别针对目的基因进行sgRNA的体外筛选，然后采用尾静脉注射敲低Pten的质粒、尾静脉注射敲低Pcsk9的AAV8病毒、眼部注射敲低Vegfa的AAV病毒。这些技术的建立为治疗人类听觉和视觉疾病提供借鉴意义。(Zhou et al. National Science Review, 2020; He et al. Protein & Cell, 2020)

    (2)神经胶质细胞转化为功能性神经元代表了一种潜在的治疗方法，用于补充与神经退行性疾病和脑损伤相关的神经元损失。使用最近开发的RNA靶向CRISPR系统CasRx的体内病毒递送，下调一个单一的RNA结合蛋白，多嘧啶束结合蛋白1（Ptbp1），能够将Müller胶质体转化为视网膜神经节细胞（RGCs），有利于RGC损失相关的疾病症状的缓解。此外，这种方法还诱导了纹状体中具有多巴胺能特征的神经元，缓解了帕金森病小鼠模型的运动缺陷。因此，由CasRx介导的Ptbp1敲除的胶质体到神经元的转换代表了一种有前途的体内遗传方法，可以作为各种神经元缺失引起的疾病治疗的潜在方案。(Zhou et al., Cell, 2020)

    代表性论文

    Gao, N., Hu. J., He, B., Ji, Z., Hu, X., Huang, J., Wei, Y., Peng, J., Wei, Y., Zhou, Y., Shen, X., Li, H., Feng, X., Xiao, Q., Shi, L., Sun, Y., Zhou, C., Zhou, H.* & Yang, H.* (2021) Endogenous promoter-driven sgRNA for monitoring the expression of low-abundant genes and lncRNAs. Nat. Cell Biol. In press.

    Ling, S., Yang, S., Hu, X., Yin, D., Dai, Y., Qian, X., Wang, D., Pan, X., Hong, J., Sun, X., Yang, H., Paludan,S. & Cai Y. * (2021) Lentiviral delivery of co-packaged Cas9 mRNA and a Vegfa-targeting guide RNA prevents wet age-related macular degeneration in mice. Nat. Biomed. Eng. In press.

    Liu, Y., Zhou, C., Huang, S., Dang, L., Wei, Y., He, J., Zhou, Y., Mao, S., Tao, W., Zhang, Y., Yang, H.*, Huang, X.* & Chi, T.* (2020) A Cas-embedding strategy for minimizing off-target effects of DNA base editors. Nat. Commun. In press.

    Fang, K., Li, Q., Wei, Y., Zhou, C., Guo, W., Shen, J., Wu, R., Ying, W., Yu, L., Zi, J., Zhang, Y., Yang, H.*, Liu, S.* & Chen, D.* (2020) Prediction and validation of mouse meiosis-essential genes based on spermatogenesis proteome dynamics. Mol. Cell Proteomics. In press.

    Zuo, E., Sun, Y., Wei, W., Yuan, T., Ying, W., Sun, H., Yuan, L., Steinmetz, L.*, Li, Y.* & Yang, H.* (2020) GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos. Nat. Protoc. 15: 3009–3029.

    Zuo, E.*, Sun, Y., Yuan, T., He, B., Zhou, C., Ying, W., Liu, J., Wei, W., Zeng, R., Li, Y.* & Yang, H.* (2020) A rationally-engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects. Nat. Methods 17: 600–604.

    Lin, X., Chen, H., Lu, Y., Hong, S., Hu, X., Gao, Y., Lai, L., Li, J., Wang, Z., Ying, W., Ma, L., Wang, N., Zuo, E.*, Yang, H.* & Chen, W.* (2020) Base editing-mediated splicing correction therapy for spinal muscular atrophy. Cell Res. 30: 548–550.

    He, B., Peng, W., Huang, J., Zhang, H., Zhou, Y., Yang, X., Liu, J., Li, Z., Xu, C., Xue, M., Yang, H.* & Huang, P.* (2020) Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver. Protein Cell 11: 518–524.

    Zhou, C., Hu, X., Tang, C., Liu, W., Wang, S., Zhou, Y., Zhao, Q., Bo, Q., Shi, L., Sun, X.*, Zhou, H.* & Yang, H.* (2020) CasRx-mediated RNA targeting prevents choroidal neovascularization in a mouse model of age-related macular degeneration. Natl. Sci. Rev. 7: 835–837.

    Zhou, H.*, Su, J., Hu, X., Zhou, C., Li, H., Chen, Z., Xiao, Q., Wang, B., Wu, W., Sun, Y., Zhou, Y., Tang, C., Liu, F., Wang, L., Feng, C., Liu, M., Li, S., Zhang, Y., Xu, H., Yao, H., Shi, L. & Yang, H.* (2020) Glia-to-neuron conversion by CRISPR-CasRx alleviates symptoms of neurological disease in mice. Cell 181: 590–603.

    Li, J., Lin, X., Tang, C., Lu, Y., Hu, X., Zuo, E., Li, H., Ying, W., Sun, Y., Lai, L., Chen, H., Guo, X., Zhang, Q., Wu, S., Zhou, C., Shen, X., Wang, Q., Lin, M., Ma, L., Wang, N., Krainer A., Shi, L.*, Yang, H.* & Chen, W.* (2020) Disruption of splicing-regulatory elements using CRISPR/Cas9 rescues spinal muscular atrophy in human iPSCs and mice. Natl. Sci. Rev. 7: 92–101.

    Yang, G., Zhou, C., Wang, R., Huang, S., Wei, Y., Yang, X., Liu, Y., Li, J., Lu, Z., Ying, W., Li, X., Jing, N., Huang, X.*, Yang, H.* & Qiao, Y.* (2019) Base-editing-mediated R17H substitution in histone H3 reveals methylation-dependent regulation of Yap signaling and early mouse embryo development. Cell Rep. 26: 302–312.

    Zuo, E., Sun, Y., Wei, W., Yuan, T., Ying, W., Sun, H., Yuan, L., Steinmetz, L.*, Li, Y.* & Yang, H.* (2019) Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364: 289–292.

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    Yao, X., Wang, X., Liu, J., Shi, L., Huang, P.* & Yang, H.* (2018) CRISPR/Cas9-mediated targeted integration In Vivo using a homology-mediated end joining-based strategy. J. Vis. Exp. 133: e56844.

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    Zhang, H., Pan, H., Zhou, C., Wei, Y., Ying, W., Li, S., Wang, G., Li, C., Ren, Y., Li, G., Ding, X., Sun, Y., Li, G., Song, L., Li, Y., Yang, H.* & Liu, Z.* (2018) Simultaneous zygotic inactivation of multiple genes in mouse through CRISPR/Cas9-mediated base editing. Development 145: dev168906.

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    Zhou, C., Zhang, M., Wei, Y., Sun, Y., Sun, Y., Pan, H., Yao, N., Zhong, W., Li, Y., Li, W.*, Yang, H.* & Chen, Z.* (2017) Highly efficient base editing in human tripronuclear zygotes. Protein Cell 8: 772–775.

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    Cheng, A., Wang, H., Yang, H., Shi, L., Katz, Y., Theunissen, T., Rangarajan, S., Shivalila, C., Dadon, D. & Jaenisch, R.* (2013) Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23: 1163–1171.

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    Gu, T., Guo, F., Yang, H., Wu, H., Xu, G., Liu, W., Xie, Z., Shi, L., He, X., Jin, S., Iqbal, K., Shi, Y., Deng, Z., Szabó, P., Pfeifer, G., Li, J.* & Xu, G.* (2011) The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature 477: 606–610.

    Yang, H., Shi, L., Zhang, S., Lin, J., Jiang, J. & Li, J. * (2010) High-efficiency somatic reprogramming induced by intact MII oocytes. Cell Res. 20: 1034-1042

    实验室网址：http://www.cebsit.cas.cn/yjz/yh_/yjfx/ 

    E-mail: huiyang@ion.ac.cn